Both biochemical and immunohistochemical techniques were used to demonstrate the presence of protein-O-carboxylmethyltransferase (PCM; E.C. 2.1.1.24) in the rodent CNS. The highest levels of immunoreactivity were detected in cortex, hippocampus, corpus striatum, thalamus and the amygdala. Other brain areas exhibited lower amounts of immunoreactive PCM. Most of the immunoreactive cells were neuronal with prominent labelling detected in both the cell body and the axonal region. Biochemical analysis of PCM activity correlated with the immunohistochemical localization. Methyl acceptor substrates for the enzyme were also high in regions rich in PCM. Western immunoblot analysis of bovine brain, rat brain and human erythrocyte forms of the enzyme showed the antisera generated against bovine brain PCM cross-reacted with human and rat forms of the enzyme, thus suggesting structural homology. These results suggest that PCM has an unique neuronal pattern of distribution, and that carboxylmethylation of proteins may be of functional significance in the nervous system.